Gene copy number variation (CNV) is ubiquitous in eukaryotic genomes and the resultant dosage changes can play an important role in determining phenotype. We aim to define the extent, evolutionary dynamics and functionality of CNV in the malaria parasite genome (Plasmodium falciparum). To achieve this we will use comparative genomic hybridization (cGH) which provides an efficient tool for assaying both CNV and single nucleotide polymorphism (SNP) and is particularly effective in haploid genomes. Preliminary data reveal abundant CNV (both deletions and amplifications) in the P. falciparum genome and demonstrate accurate prediction of SNPs. The project will be divided into three parts. First, we will identify all CNV and catalogue flanking SNPs in a worldwide parasite collection. The populations sampled will comprise freshly isolated single-clone infections from Thailand, Papua New Guinea, Colombia and Gambia. We will measure the size, gene content, and arrangement and population frequency of genome rearrangements. Our second aim will be to evaluate evidence for adaptive evolution of genome regions showing CNV to highlight genome segments that could be influenced by recent evolutionary pressures such as selection by drugs. We will do this by examining patterns of linkage disequilibrium and haplotype structure in flanking SNP polymorphism, geographical variation in frequency, and by examination of numbers of independent origins of CNVs. Finally, we will conduct focused investigations of one CNV - containing the GTP cyclohydrolase locus - that is likely to be of functional importance. We will test the hypothesis that amplification of this gene compensates for reduced metabolic efficiency of enzymes further down the folate pathway carrying mutations that confer resistance to anti-folate compounds. This will be done using selection and transfection experiments in combination with assays of drug resistance, fitness, and flux through the folate pathway. Relevance of this research to public health. Variation in the number of gene copies can have a large influence on parasite biology. We propose to systematically describe DNA rearrangements in the malaria parasite genome, and evaluate the role that they play in this important pathogen.